Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

Current status of pharmacogenomics testing for anti-tumor drug therapies : approaches to non-melanoma skin cancer

Skin cancer is one of the most commonly occurring cancer types, with substantial social, physical, and financial burdens on both individuals and societies. Although the role of UV light in initiating skin cancer development has been well characterized, genetic studies continue to show that predisposing factors can influence an individual's susceptibility to skin cancer and response to treatment. In the future, it is hoped that genetic profiles, comprising a number of genetic markers collectively involved in skin cancer susceptibility and response to treatment or prognosis, will aid in more accurately informing practitioners' choices of treatment. Individualized treatment based on these profiles has the potential to increase the efficacy of treatments, saving both time and money for the patient by avoiding the need for extensive or repeated treatment. Increased treatment responses may in turn prevent recurrence of skin cancers, reducing the burden of this disease on society. Currently existing pharmacogenomic tests, such as those that assess variation in the metabolism of the anticancer drug fluorouracil, have the potential to reduce the toxic effects of anti-tumor drugs used in the treatment of non-melanoma skin cancer (NMSC) by determining individualized appropriate dosage. If the savings generated by reducing adverse events negate the costs of developing these tests, pharmacogenomic testing may increasingly inform personalized NMSC treatment.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation ‘‘hotspot’’ at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

Analysis of acute-phase proteins, AHSG, C3, CLI, HP and SAA, reveals distinctive expression patterns associated with breast, colorectal and lung cancer

Early detection, clinical management and disease recurrence monitoring are critical areas in cancer treatment in which specific biomarker panels are likely to be very important in each of these key areas. We have previously demonstrated that levels of alpha-2-heremans-schmid-glycoprotein (AHSG), complement component C3 (C3), clusterin (CLI), haptoglobin (HP) and serum amyloid A (SAA) are significantly altered in serum from patients with squamous cell carcinoma of the lung. Here, we report the abundance levels for these proteins in serum samples from patients with advanced breast cancer, colorectal cancer (CRC) and lung cancer compared to healthy controls (age and gender matched) using commercially available enzyme-linked immunosorbent assay kits. Logistic regression (LR) models were fitted to the resulting data, and the classification ability of the proteins was evaluated using receiver-operating characteristic curve and leave-one-out cross-validation (LOOCV). The most accurate individual candidate biomarkers were C3 for breast cancer [area under the curve (AUC) = 0.89, LOOCV = 73%], CLI for CRC (AUC = 0.98, LOOCV = 90%), HP for small cell lung carcinoma (AUC = 0.97, LOOCV = 88%), C3 for lung adenocarcinoma (AUC = 0.94, LOOCV = 89%) and HP for squamous cell carcinoma of the lung (AUC = 0.94, LOOCV = 87%). The best dual combination of biomarkers using LR analysis were found to be AHSG + C3 (AUC = 0.91, LOOCV = 83%) for breast cancer, CLI + HP (AUC = 0.98, LOOCV = 92%) for CRC, C3 + SAA (AUC = 0.97, LOOCV = 91%) for small cell lung carcinoma and HP + SAA for both adenocarcinoma (AUC = 0.98, LOOCV = 96%) and squamous cell carcinoma of the lung (AUC = 0.98, LOOCV = 84%). The high AUC values reported here indicated that these candidate biomarkers have the potential to discriminate accurately between control and cancer groups both individually and in combination with other proteins. Copyright © 2011 UICC.

Tumor necrosis correlates with angiogenesis and is a predictor of poor prognosis in malignant mesothelioma

Objectives: Malignant mesothelioma (MM) is a fatal tumor of increasing incidence related to asbestos exposure. Microscopic tumor necrosis (TN) is a poor prognostic factor in solid tumors, but it has not been characterized in MM. We wished to evaluate the incidence of TN in MM and its correlations with clinicopathologic factors, angiogenesis, and survival. Methods: TN was graded in 171 routine formalin-fixed, paraffin-embedded hematoxylin-eosinstained tumor sections by two independent observers. Angiogenesis was assessed by the microvessel count (MVC) of CD34 immunostained sections. TN was correlated with survival by Kaplan-Meier and log-rank analysis, and stepwise, multivariate Cox models were used to compare TN with angiogenesis and established prognostic factors and prognostic scoring systems. Results: TN was identified in 39 cases (22.8%) and correlated with low hemoglobin (p = 0.01), thrombocytosis (p = 0.04), and high MVC (p = 0.02). TN was a poor prognostic factor in univariate analysis (p = 0.008). Patients with TN had a median survival of 5.3 months vs 8.3 months in negative cases. Independent indicators of poor prognosis in multivariate analysis were nonepithelioid cell type (p = 0.0001), performance status > 0 (p = 0.007), and increasing MVC (p = 0.004) but not TN. TN contributed independently to the European Organisation for Research and Treatment of Cancer (EORTC) [p = 0.03] and to the Cancer and Leukemia Group B (CALGB) [p = 0.03] prognostic groups in respective multivariate Cox analyses. Conclusions: TN correlates with angiogenesis and is a poor prognostic factor in MM. TN contributes to the EORTC and CALGB prognostic scoring systems.

Advances in the systemic therapy of malignant pleural mesothelioma

Malignant pleural mesothelioma is an aggressive thoracic malignancy associated with exposure to asbestos, and its incidence is anticipated to increase during the first half of this century. Chemotherapy is the mainstay of treatment, yet sufficiently robust evidence to substantiate the current standard of care has emerged only in the past 5 years. This Review summarizes the evidence supporting the clinical activity of chemotherapy, discusses the use of end points for its assessment and examines the influence of clinical and biochemical prognostic factors on the natural history of malignant pleural mesothelioma. Early-phase clinical trials of second-line and novel agents are emerging from an increased understanding of mesothelioma cell biology. Coupled with high-quality translational research, such developments have real potential to improve the outlook of patients at a time of increasing incidence.

Overexpression of the mammalian target of rapamycin (mTOR) and angioinvasion are poor prognostic factors in early stage NSCLC: A verification study

Background: A recent study by Dhillon et al. [12], identified both angioinvasion and mTOR as prognostic biomarkers for poor survival in early stage NSCLC. The aim of this study was to verify the above study by examining the angioinvasion and mTOR expression profile in a cohort of early stage NSCLC patients and correlate the results to patient clinico-pathological data and survival. Methods: Angioinvasion was routinely recorded by the pathologist at the initial assessment of the tumor following resection. mTOR was evaluated in 141 early stage (IA-IIB) NSCLC patients (67 - squamous; 60 - adenocarcinoma; 14 - others) using immunohistochemistry (IHC) analysis with an immunohistochemical score (IHS) calculated (% positive cells × staining intensity). Intensity was scored as follows: 0 (negative); 1+ (weak); 2+ (moderate); 3+ (strong). The range of scores was 0-300. Based on the previous study a cut-off score of 30 was used to define positive versus negative patients. The impact of angioinvasion and mTOR expression on prognosis was then evaluated. Results: 101 of the 141 tumors studied expressed mTOR. There was no difference in mTOR expression between squamous cell carcinoma and adenocarcinoma. Angioinvasion (p= 0.024) and mTOR staining (p= 0.048) were significant univariate predictors of poor survival. Both remained significant after multivariate analysis (p= 0.037 and p= 0.020, respectively). Conclusions: Our findings verify angioinvasion and mTOR expression as new biomarkers for poor outcome in patients with early stage NSCLC. mTOR expressing patients may benefit from novel therapies targeting the mTOR survival pathway. © 2011 Elsevier Ireland Ltd.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers

Background: To directly assess tumor oxygenation in resectable non - small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes. Methods: Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes. Results: The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO 2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001). Conclusions: Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors. © 2006 American Association for Cancer Research.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE:: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation. Copyright © 2009 by Lippincott Williams & Wilkins.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Angiogenesis as a biomarker and target in cancer chemoprevention

Angiogenesis, the formation of new blood vessels from existing vasculature, is essential to the late stages of carcinogenesis, allowing tumours to grow beyond 1-2 mm in diameter, invade surrounding tissue, and metastasise. However, more than two decades ago, angiogenesis that preceded neoplastic transformation was seen. Indeed, it can be detected in inflammatory and infectious diseases that increase the risk of developing cancer. Recent advances in fluorescence endoscopy and histological assessment suggest that, for certain cancers, the degree of new blood-vessel formation may differ between the early and late stages of carcinogenic progression. The association between angiogenesis and cancer occurrence, and ease of detection of this process in accessible tissues early in carcinogenesis, mean that angiogenesis fulfils the criteria for a biomarker of the effectiveness of chemopreventive intervention. There is also some evidence that biochemical assays of angiogenic growth factors may after similar potential as surrogate biomarkers. Many natural and synthetic chemopreventive agents in development or in clinical use inhibit new vessel formation in vivo. Validation of angiogenesis as a biomarker for the effectiveness of chemoprevention should further the advancement of some chemopreventive agents.